Communicated by M. A. Saghai Maroof

T. Barreneche · C. Bodenes · R. Streiff · C. Plomion · G. Roussel ·A. Kremer ( )Laboratoire de Genetique et d’Amelioration des Arbres Forestiers,INRA, B.P. 45, Pierroton, F-33610 Cestas Cedex, FranceFax: (33) 5 57 97 90 88E-mail: kremer@pierroton.inra.fr

C. Lexer · J. Glossl · H. SteinkellnerZentrum fur Angewandte Genetik, Universitat fur BodenkulturWien, Muthgasse 18, A-1190 Vienna, Austria

J.-F. Trontin · J.-M. FavreLaboratoire de Biologie Forestiere, associe INRA, Universite HenriPoincare, Nancy, B.P. 239, 54506 Vandoeuvre-les-Nancy, France

S. Fluch · K. BurgForschungszentrum Seibersdorf, A-2444 Seibersdorf, Austria

Theor Appl Genet (1998) 97 : 1090—1103 ( Springer-Verlag 1998

T. Barreneche · C. Bodenes · C. Lexer · J.-F. TrontinS. Fluch · R. Streiff · C. Plomion · G. Roussel · H. SteinkellnerK. Burg · J.-M. Favre · J. Glossl · A. Kremer

A genetic linkage map of Quercus robur L. (pedunculate oak)based on RAPD, SCAR, microsatellite, minisatellite, isozymeand 5S rDNA markers

Received: 30 January 1998 / Accepted: 12 May 1998

Abstract A genetic map of Pedunculate oak (Quercusrobur) was constructed based on one 5S rDNA, 271RAPD, ten SCAR, 18 microsatellite, one minisatellite,and six isozyme markers. A total of 94 individuals froma full-sib family was genotyped. Two maps, including307 markers, were constructed according to the ‘‘two-way pseudo-testcross’’ mapping strategy. Testcrossmarkers segregating in the 1 : 1 ratio were first used toestablish separate maternal (893.2 cM, 12 linkagegroups) and paternal (921.7 cM, 12 linkage groups)maps. Both maps provided 85—90% genome coverage.Homologies between the male and female linkagegroups were then identified based on 74 intercrossmarkers segregating in the 3 : 1, 1 : 2 : 1 and 1 : 1 : 1 : 1ratios (RAPDs, SCARs, SSRs, 5S rDNA and isozymes)in the hybrid progeny. In each map, approximately18% of the studied markers showed segregation distor-tion. More than 60% of the skewed markers were dueto an excess of heterozygote genotypes. This map willbe used for: (1) studying the molecular organisation ofgenomic regions involved in inter- and intraspecificdifferentiation in oaks and (2) identification of QTLsfor adaptive traits.

Key words Quercus robur L · Linkage map · RAPD ·SCAR · Microsatellite · Minisatellite · 5S rDNA ·Isozymes

Introduction

Depending on the authors the number of existing oakspecies varies between 300 and 600. They are spreadover the northern hemisphere (Europe, Africa, NorthAmerica and Asia) from boreal zones to semi-aridareas. Pedunculate oak (Quercus robur L.) is one of themost important and widespread oak species in Euro-pe. It belongs to the white oak section (¸epidobalanus)and is distributed throughout Europe from centralSpain to the Urals. According to Camus (1954) the¸epidobalanus section is the richest with respect tonumbers and diversity and comprises more than 150species spread over all four continents.

Quercus robur L. has a broad ecological range, fromvery acid to chalky and from moist to mesophilic soils,but it prefers to grow on neutral and moist soils(Becker and Levy 1990). Genetic studies in peduncu-late oak have mostly concentrated on populationgenetics, provenance research and the inheritance ofeconomically important traits (Kremer et al. 1993, fora review).

Cytogenetic investigations have been restricted tokaryotypic analysis. Quercus robur L. is a diploid spe-cies (2n"2x"24), although in some cases super-numerary chromosomes (B type) have been reported(Ohri and Ahuja 1990; Besendorfer et al. 1996). Natu-ral triploid individuals have been described (Johnsson1946; Butorina 1993) that usually are larger than di-ploid trees. Compared to data available on otherwoody angiosperms (e.g. Populus, Eucalyptus, Acacia,Pyrus, Fraxinus) the physical genome size of Q. roburis larger (1.88 pg/2C) (Zoldos\ et al. 1998), although itssize is among the lower values found in plants (Favre

and Arnould 1996; Favre and Brown 1996). Segrega-tion studies with gene markers have been conductedin Quercus robur with isozymes (Zanetto et al.1996), SCARs (sequence characterised amplified re-gions, Bodenes et al. 1996) and microsatellites (Stein-kellner et al. 1997a). However, due to the smallnumber of loci analysed in each of these studies, nolinkage groups could be constructed except withisozymes when two linkage groups were identified(Zanetto et al. 1996).

Our objective was to construct a genetic map withall earlier developed markers (isozymes, SCARs,microsatellites, minisatellites, 5S rDNA). In order tolocate these markers in the genome, we added ran-domly distributed markers using amplification witharbitrarily designed primers [random amplified poly-morphic DNA (RAPD) Williams et al. 1990]. Thepedigree used for the map construction is a full-sibF1

progeny of Quercus robur L. Linkage groups wereassembled according to the ‘‘two-way pseudo-test-cross’’ method described by Grattapaglia and Sederoff(1994).

To our knowledge, this is the first reported linkagemap in the genus Quercus, and in the Fagaceae familyas well. Some of the markers used in the construction ofthe map have been reported to be applicable to otheroak species: isozymes in Q. petraea and Q pubescens(Muller-Starck et al. 1996), microsatellites in other sec-tions of the genus Quercus (Steinkellner et al. 1997b).Because oaks of a given botanical section usually ex-hibit high genetic similarities (Kremer and Petit 1993),we expect that the present map will provide a usefultool for various applications in population and quant-itative genetics in different white oak species.

Materials and methods

Plant material

Segregation data from a two-generation full-sib pedigree including94 individuals were used to construct two genetic maps, one for eachparental tree. The male parent originated from near Arcachon(southwest of France), and the female parent was located on theForestry Research Station in Pierroton (southwest of France). Thecontrolled cross was made in the spring of 1992 according to thetechniques described in Zanetto et al. (1996). About 400 acorns werecollected in pollination bags, of which 94 were used for map con-struction. Preliminary segregation analysis of the 94 offspring witha few isozymic loci did not detect any acorn resulting from fertilisa-tion with foreign pollen (Zanetto et al. 1996).

Marker analysis

Genomic DNA was extracted from leaves according to Saghai-Maroof et al. (1984) with some minor modifications described byMoreau et al. (1994).

Procedures used to study different markers are summarized inTable 1. Specific primer sequences and annealing temperatures for

the SCAR, microsatellite, minisatellite and 5S rDNA markers areindicated in Table 2.

Data analysis

After segregation was scored for each marker, Mendelian segrega-tion was tested in the offspring using Chi-square goodness-of-fittests. Linkage analysis of the markers was performed using a combi-nation of MAPMAKER (Lander et al. 1987) and JOINMAP (Stam1993) software (see Results section). The Kosambi (Kosambi 1944)mapping function was used to convert recombination frequencies tomap distances in centiMorgans.

Genome length, E(G), of both parental maps was estimated underthe assumption of random marker distribution according to method‘‘3’’ of Chakravarti et al. (1991): G"2MX/K, where M"n(n!1)/2is the number of informative meioses (n is the number of locianalysed), X is the maximum observed map distance among thelocus pairs above a threshold LOD, Z, and K is the number of locuspairs having LOD values at or above Z. The values tested for Z were3, 4 and 5. The values of X and K were obtained using the ‘‘LODs’’function of MAPMAKER with the maximum recombination frac-tion set to 0.5 and minimum LOD threshold set to Z. Values forX were obtained using the Kosambi map function. These estima-tions were calculated by taking into consideration only pairwisecomparisons between framework markers.

Results

Segregation analysis

Markers were subdivided in four different groups onthe basis of the segregation patterns in the progeny:

A) Loci that were in the testcross configuration (Grat-tapaglia and Sederoff 1994) between the parents(heterozygous in one parent and homozygous nullin the other, or vice-versa). A 1 : 1 segregation ratiowas observed in the F

1family for most RAPDs,

seven SCARs (six dominant and one codominant),two isozymes, 1 microsatellite (ssrQpZAG104) andone minisatellite (Omin3).

B) Loci that were heterozygous in both parents andthat segregated in a 3 : 1 ratio in the offsprings. FiftyRAPDs followed this pattern.

C) Loci that were heterozygous in both parents andthat segregated in a 1 : 2 : 1 ratio. This was the casefor 2 microsatellites and three isozymes.

D) Loci that were heterozygous in both parents andthat segregated in a 1 : 1 : 1: 1 ratio. Three SCARs,15 microsatellites, one isozyme and one 5S rDNAlocus followed this pattern. Markers segregating inthe 1 : 1 : 1 : 1 ratio were partitioned according to thealleles contributed by the relevant parent (Jacobs etal. 1995), resulting in a marker segregating 1 : 1 inboth the female and the male parent, as for categoryA markers.

Approximately 80% of the markers analysed in theprogeny fit the expected Mendelian segregation ratios(1 : 1, 3 : 1, 1 : 2 : 1 or 1 : 1 : 1 : 1). A total of 61 markersshowed segregation distortion at a"0.05.

1091

Table 1 Protocols used for the molecular analysis of the different markers

Marker type Number of PCR protocol Electrophoresis Staining Referencesmarkers conditions

Primer Amplification conditionstype

RAPD 271 Ten-baseprimersOperon

1 step 94°C 4 min 1.8% agarose gel Ethidiumbromide

Williams et al. 1990

Technologieskits A-Z 92°C 45 s

35 cycles 40°C 45 s72°C 1 min 45 s

SCAR 10 * 1 step 94°C 3 min 1% agarose gel Ethidium Bodenes et al. 199794°C 45 s or bromide

30 cycles T°C 45 s 8% non-denaturating Silver staining72°C 1 min 30 s polyacrylamide gel

Microsatellite 18 * 1 step 95°C 6 min 6% denaturatingpolyacrylamide gel

Fluorescence(ALF express)

Steinkellner et al.1997b

T°C 1 min30 cycles 72°C 30 s Silver staining Streiff et al. 1998

92°C 1 min1 step 72°C 8 min

Minisatellite 1 * 1 step 94°C 4 min 2% agarose gel Ethidium Fluch and Burg94°C 45 s bromide unpublished data

30 cycles 50°C 45 s72°C 30 s

5S rDNA 1 * 1 step 96°C 3 min 8% non-denaturing Silver staining Specht et al. 199794°C 40 s polyacrylamide gel

30 cycles 55°C 1 min72°C 40 s

Isozymes 6 — — 11%—12% starchgel or IEF

" Zanetto et al. 1996

! See Table 2 for specific primer sequences and annealing temperatures"See Zanetto et al. (1996) for staining conditions of various enzyme systems

1092

Table 2 Primer pairs and annealing temperatures of SCAR, microsatellite, minisatellite and 3Sr DNA markers

Locus name Primer sequence Annealing Referencestemperature (°C)

A17-700 F: GAC CGC TTG TTG GTA 55 Bodenes et al. 1997B: GAC CGC TTG TGA TTA

F: AGA CCC GTA GAG GAG ACA TT Bodenes et al. 1997B11-1500B: CCC GTG TAG TAT TCC ACA AA

52

F: GTC TTC GAC TGG GGT GAA Bodenes et al. 1997B12-500B: TCA GTC CAT TCC GAA AGA

52

F: CCT TGA ACG CAT TATGAC AT Bodenes et al. 1997B12-800B: CCT TGA ACG CAG CAC AAT TC

48 Bodenes et al. 1997

F: CAG AAG AAG CAA TGG TAA CAF14-750B: CAA CAT TTG GTG TGT CTT AG

50 Bodenes et al. 1997

F: GTG TGG TGC AGA AAAI13-300B: ACT CCA GGT CTA TCC

52 Bodenes et al. 1997

F: TGC AAA ACATAA ACA TTP14-450B: AAT CCA GTG GCA AGT TTT AA

48 Bodenes et al. 1997

F: CGC ATG GAT ATA CTA TTCP17-1400B: CTA CCT AGT GTCCTA TGG

54 Bodenes et al. 1997

F: CGA GCG TTG ATA GCC AAT AGR12-500B: TCG GAA GCA AAA GGG TAA TT

51 Bodenes et al. 1997

F: GCC CTA ACA AAT CAT CTCU7-790B: TAA TAT AGA AAG GGA AAG

48 Steinkellner et al. 1997a

F: GCT TGA GAG TTG AGA TTT GTssrQpZAG1/5B: GCA ACA CCC TTT AAC TAC CA

55 Steinkellner et al. 1997a

F: CTT CAC TGG CTT TTC CTC CTssrQpZAG16B: TGA AGC CCT TGT CAA CAT GC

59 Steinkellner et al. 1997a

F: TAG AAA GCC CAA AAC CAA AAC CssrQpZAG3/64B: CTT TTT GGA AGC CGC TTC CGT A

50 Steinkellner et al. 1997a

F: CTT GAG CAT GGA ATC CTA TGssrQpZAG3/62B: TCT AGA GGA GCT TTC CTT TAC AC

58 Steinkellner et al. 1997a

F: GCA ATT ACA GGC TAG GCT GGssrQpZAG9B: GTC TGG ACC TAG CCC TCA TG

50 Steinkellner et al. 1997a

F: CGA TTT GAT AAT GAC ACT ATG GssrQpZAG15B: CAT CGA CTC ATT GTT AAG CAC

50 Steinkellner et al. 1997a

F: GAT CAA ATT TGG AAT ATT AAG AGA GssrQpZAG36B: ACT GTG GTG GTA GTC TAA CAT GTA G

50 Steinkellner et al. 1997a

F: CCC CTA TTG AAG TCC TAG CCGssrQpZAG46B: TCT CCC ATG TAA GTA GCT CTG

48 Steinkellner et al. 1997a

F: CTG CAA GAT TCG GAC AAG CAAssrQpZAG58B: TCT TTT TTC CTA ATC TCA CCT G

50 Steinkellner et al. 1997a

F: CAA CAA TGA AAG GGA GAA AGCssrQpZAG102B: CGC TTT TGA CTT TCC TAC CTT

50 Steinkellner et al. 1997a

F: ATA GGG AGT GAG GAC TGA ATGssrQpZAG104B: GAT GGT ACA GTC GCA ACA TTC

50 Steinkellner et al. 1997a

F: CTA GCC ACA ATT CAG GAA CAGssrQpZAG108B: CCT CTT TTG TGA ATG ACC AAG

50 Steinkellner et al. 1997a

F: GGA GGC TTC CTT CAA CCT ACTssrQpZAG110B: GAT CTC TTG TGT GCT GTA TTT

48 Steinkellner et al. 1997a

F: GAT CAG TGA TAG TGC CTC TCssrQpZAG119B: GAT CAA CAA GCC CAA GGC AC

50 Steinkellner et al. 1997a

F: TCT CCT CTC CCC ATA AAC AGGMSQ4B: GTT CCT CTA TCC AAT CAG TAG TGA G

50 Dow et al. 1995

1093

Table 2 Continued

Locus name Primer sequence Annealing Referencestemperature (°C)

F: TGG CTG CAC CTA TGG CTC TTA GMSQ13B: ACA CTC AGA CCC ACC ATT TTT CC

50 Dow et al 1995

F: GGA ACA ACT AGA GAG AAC CAA GTC AGGMSQ16B: TTG CCT ATC CTG CC CCG TAT CAC

50 Dow and Ashley 1996

F: CGA TTT TGA ACA CCT TCT TCMicJ-AG22B: CGA TGT GGG ATT TTG ATT TT

46 T. Kawahara (personal

F: AGA ATC GAC TTC AAT GCA AG

communication)

Omin3B: CCA TTC ACA GAG GTT GTC GG

50 Fluch et al. (personal

F: TGG GAA GTC CTC GTG TTG CA

communication)

5S rDNAB: ATT AGT GCT GGT ATG ATC GC

55 Specht et al. 1997

c

Fig. 1 Framework linkage maps of Quercus robur L. Female map onthe left and male map on the right. Markers were mapped witha minimal LOD score of 3.0. Accessory markers (testcross andintercross types) were located beside their closest framework marker.Intercross markers (category B and C, see Results section) and somerecoded testcross markers (category D) were used to identify homo-logous linkage groups. These markers are interconnected with boldlines. Intercross RAPD markers are boxed, and microsatellite, mini-satellite, SCAR and 5S rDNA markers are circled. Markers display-ing distorted segregation (a"0.05) are denoted by boldface asterisk

Map construction

The map construction was done in two steps. First, twoseparate parental maps (paternal and maternal) wereconstructed using markers segregating in the 1 : 1 ratio(category A and D markers) with a confidence level ofP40.01. These ‘‘testcross’’ markers were grouped withthe help of MAPMAKER (Lander et al. 1987) using thefollowing criteria:

— a minimum LOD score of 3.0 for statistical accept-ance of linkage.

— a maximum recombination fraction h of 0.40.

In the initial step of map construction, the order ofnon-distorted markers was approximated using the‘‘FIRST ORDER’’ command. From this order, a subsetof markers that could be locally ordered with a likeli-hood ratio greater than LOD"2 was used to estab-lish a framework map using the ‘‘RIPPLE’’ command.Markers that could not meet this ordering criteria weredropped from the framework and placed as accessorymarkers. Accessory markers were then located besidetheir closest framework marker. Distorted markers thatwere excluded from the framework map were alsoplaced in the established framework map as accessorymarkers using the ‘‘NEAR’’ command. These markersare indicated with a boldface asterisk in Fig. 1.

In a second step, intercross markers segregating inboth parents (category B, C) in addition to categoryD markers already mapped in the first step were usedto identify homologous counterparts between parentalmaps. Markers of category D were mapped usingMAPMAKER. However, because MAPMAKER doesnot accept mixed segregation data, intercross dominantand codominant markers of category B and C wereplaced to an approximate location on both maps withthe JOINMAP programme (Stam 1993).

Finally, a total of 307 Loci (271 RAPDs, ten SCARs,18 microsatellites, one minisatellite, six isozymes andone 5S rDNA marker) were located on both oak link-

age maps (Fig. 1). Among the 50 intercross RAPD loci(category B) only the 24 markers showing strong link-age (LOD54.5) with framework markers in bothmaps were retained.

Female map (Fig. 1)

A total of 136 markers were used to establish the femalemap. Approximately 58% of them (79 loci) were lo-cated on the framework map. Twelve linkage groupswere set up for the female map covering 893.2 cM, withan average distance between 2 framework loci of11.30 cM. The size of the linkage groups ranged from33 cM to 124.8 cM, with an average of 74.40$16.90 cM.

Male map (Fig. 1)

The male map had 136 markers with 83 loci (60%)establishing the framework and covering 921.7 cM. Theaverage distance between 2 framework loci was11.10 cM. The framework loci were mapped into 12linkage groups. The length of the groups ranged be-tween 9.1 cM and 191.8 cM, with an average of70.90$27.80 cM.

Fifty-two markers in the female map and 44 markersin the male map (testcross loci), including RAPDs,

1094

1095

Fig. 1 Continued. (See page 1094 for legend)

1096

Fig. 1 Continued. (See page 1094 for legend)

1097

SSRs, isozymes and SCARs, were used as accessorymarkers.

The number of linkage groups corresponded to thenumber of haploid chromosomes of Quercus: n"12, inboth maps.

Homology between parental maps

Markers segregating in both parents including inter-cross markers (50 RAPDs, three isozymes and 2 micro-satellites) and loci segregating 1 : 1 : 1 : 1 (three SCARs,15 microsatellites, one isozyme and one 5S rDNAmarker) were used to identify homology between par-ental maps. These ‘‘bridge’’ markers are connected bybold lines in Fig. 1. Hence, ten homologous linkagegroups were identified.

Because of low information content between marker-pairs corresponding to category A, B and A, C (Ritteret al. 1990), the use of codominant markers segregating1 : 1 : 1 : 1 and therefore segregating 1 : 1 from both par-ents is a prerequisite for constructing an accurate com-bined map. Merging the male and female maps was notour primary goal but this would be possible if moremicrosatellites were available.

Segregation distortion

At a"0.05, segregation distortions from the Men-delian expectation of testcross markers (category A andD markers) were detected at 25 marker loci in thefemale map and 24 marker loci in the male map. Thedistortion level was quite similar in both parents (18%).At a"0.01, the number of distorted markers decreasedto 13 in the female map and 11 in the male map. Sixtypercent of category A-distorted RAPD markers in themale map (66% in the female) displayed an excessheterozygote genotypes.

Fifteen intercross RAPD markers showed strong de-viations from Mendelian expectations, but only 2 ofthese were associated with a LOD score 54.5 toa framework marker in the map. Finally, a total of 61markers (20%) showed segregation distortions ata"0.05. This distortion level is higher than expectedby chance alone (15 distorted markers at a"0.05).Markers exhibiting segregation distortion were notrandomly distributed in the genome. They tended to bepreferentially grouped in a few clusters as shown onFig. 1.

Genome length and map coverage

The estimated total map length (average of threevalues) for the female parent amounted to E(G)"1192 cM and for the male parent to E(G)"1235 cM.These values are the first to be reported for oak. The

different estimates, obtained from a range of Z values,were very close (data not shown) and were higher thanthe observed genome length of 893.2 cM for the femaleand 921.7 cM for the male map. This indicated that themapped loci did not provide full coverage of the oakgenome. Although the number of linkage groups cor-responded to the number of haploid chromosomes(n"12), 2 small linkage groups (LG12) were identifiedfor both parental maps. In addition, linkage group 1 ofthe female map was homologous to 2 unlinked linkagegroups (1a and 1b) of the male map. This gap could befilled in by additional markers. Following Lange andBoehnke (1982), the minimal number, N, of randomlydistributed markers required to cover a proportionof P"95% of a genome of size of L"1200 cM at amaximum distance between markers of 2d"20 cM is:N"ln(1!p)/ln(1!2d/L)"178. This expectationwas higher than the number of markers actually used inthe initial step of map construction (136 markers forboth the male and female map). According to thisformula, 136 markers would provide 90% coverage fora 20-cM map, a percentage coverage that was veryclose to that obtained following Bishop et al. (1983)(85% for both maps).

The assumption of a random distribution of markersin the genome seemed to fit our experimental datamainly based on RAPDs. The observed and expecteddistributions of markers were compared for 20-cMintervals. A chi-square test for departure from a Pois-son distribution was computed for six classes or groupsof classes containing at least five observations. Themean of the Poisson distribution was set to the meannumber of markers per 20-cM interval length. No sig-nificant departure (a"0.01) from the Poisson expecta-tion was observed in this goodness-of-fit test. A randomdistribution of RAPD markers was also observed inmaritime pine (Plomion et al. 1995).

Discussion

Pseudo-test-cross mapping in Q. robur

The Q. robur maps presented here were constructedfollowing the ‘‘two-way pseudo-testcross’’ strategy.Two maps of similar size (893 cM in the female parent,922 cM in the male parent) were obtained for eachparent, each one represented by 12 linkage groupscorresponding to the 12 haploid chromosomes identi-fied in oaks (Ohri and Ahuja 1990). The two maps arefurthermore equally balanced in number of frameworkmarkers, mean size of linkage groups and mean dis-tance between framework markers. As oaks exhibithigh levels of heterozygozity in natural populations(Kremer and Petit 1993; Zanetto et al. 1994), 18% ofthe RAPD markers were found to be heterozygous inboth parents (these are referred to as intercross locisegregating in the 3 : 1 ratio) compared to the 82% that

1098

Table 3 Map information on some woody angiosperms and annual plants

Species Population Number Marker Number Observed Map Estimated References(chromosome type of maps type of markers map length length map length"

number) coverage((pg/C))

Citrus Intergeneric BC1

1 RFLP 69 553 cM — 900—1350 cM Durham et al. 1994(2n"18) isozymes((0.62 pg/C)) Intergeneric BC

11 RAPD, RFLP 312 1192 cM 70—80% Cal et al. 1994

Intergeneric BC1

2 RAPD, RFLP 100 633.1 cM (female) — Luro et al. 1995isozymes 1503 cM (male) —

Intergeneric F1

1 RFLP, SSR 56 410 cM — Kijas et al. 1997isozymes

Eucalyptus Interspecific F1

2 RAPD 491 1552 cM (female) 95.80% 1100—1650 cM Grattapaglia and Sederoff 1994(2n"22) 1101 cM (male) 95.20%((0, 6 pg/C)) Interspecific F

12 RAPD 505 1331 cM (female) 95—99% Verhaegen and Plomion 1996

1415 cM (male) 95—99%Intraspecific G

21 RAPD, RFLP

isozymes339 1462 cM — Byrne et al. 1995

Persea Interspecific F1

2 RAPD, SSR 90 352.6 cM — 1200—1800 cM Sharon et al. 1997(2n"24) minisatellites((0, 9 pg/C))

Malus F1

1 RAPD, RFLP 409 950 cM — 1700—2550 cM Hemmat et al. 1994(2n"34) isozymes((0.8 pg/C))

Populus Interspecific F1

1 RAPD, RFLP 343 1261 cM 48.50% 1900—2850 cM Bradshaw et al. 1994(2n"38) isozymes, STS((0.6 pg/C))

Prunus(2n"16)

Interspecific F2

1 RFLP, isozymes,morphological

118 800 cM — 800—1200 cM Foolad et al. 1995

((0.3 pg/C))

Prunus persica Interspecific F2

1 RAPD, RFLP 65 332 cM (50% Rajapakse et al. 1995Intraspecific F

21 RAPD, isozymes, 99 396 cM — Chaparro et al. 1994

morphologicalIntraspecific F

21 RFLP, RAPD, 270 712 cM &100% Dirlewanger et al. 1998

AFLP, IMA,morphological

Prunus amygdalus Intraspecific F1

2 RFLP, 127 393 cM (female) — Viruel et al. 1995isozymes 394 cM (male)

Quercus robur L.(2n"24)((0.9 pg/DC))

Intraspecific F1

2 RAPD, SCAR,SSR, isozymes5S rDNA

307 893.2 cM (female) 85% 1200—1800 cM Barreneche et al. 1998(this study)

921.7 cM (male) 85%

1099

Table 3 Continued

Species Population Number Marker Number Observed Map Estimated References(chromosome type of maps type of markers map length length map length"

number) coverage((pg/C))

¹heobroma cacao L.(2n"20)

Intraspecific F1

1 RAPD, RFLP,isozymes

193 759 cM — 1000—1500 cM Lanaud et al. 1995(this study)

((0.4pg/C))Intraspecific BC

11 RAPD, RFLP 138 1068 cM — Crouzillat et al. 1996

»itis Interspecific F1

2 RAPD, RFLP, 523 1196 cM (female) — 1900—2850 cM Lodhi et al. 1995(2n"38) isozymes((0.5 pg/C)) 1477 cM (male) —

Arabidopsis thaliana(2n"10)((0.15 pg/C))

RIL 1 RAPD,microsatellites

302 675 cM &100% 500—750 cM Reiter et al. 1992

30 Bell et al. 1994#

Hordeum vulgare(2n"14)((5.50 pg/C))

DH 1 RAPD, RFLP,isozymes

295 1250 cM &100% 700—1050 cM Kleinhofs et al. 1993

¸ycopersiconesculentum

F2

1 RFLP, isozymes,morphological

1030 1276 cM &100% 1200—1800 cM Tanksley et al. 1992

(2n"24)((0.98 pg/C))

Zea mays(2n"20)((2.5 pg/C))

RIL, F2

1 RFLP, isozymes,molecularmarkers

275 1765 cM &100% 1000—1500 cM Causse et al. 1996

! F1, Two-generation outbred pedigree; F

2, three-generation inbred pedigree; G

2, three-generation outbred pedigree, RIL, recombinant inbred lines; DH, doubled-haploid lines; BC

1,

first-generation backcross" 100—150 cM per chromosome according to Rajapakse et al. (1995)# See http://cbil.humgen.upenn.edu/Natgc/SSLP-info/SSLP-map.html

1100

segregated in a testcross configuration. Reported cor-responding values in other species using the‘‘two-waypseudo-testcross’’ are quite different: the number ofRAPDs segregating in 3 : 1 proportions varied between2% and 5% in Eucalyptus studies (Grattapaglia andSederoff 1994; Verhaegen and Plomion 1996) andamounted to 5% in pines (Kubisiak et al. 1995). Thesedifferences can likely be imputed to the differences inlevels of heterozygozity between these species. Thehigher proportion of heterozygote RAPD loci in bothparents also allowed the assignment of homologouslinkage groups of the two parents as suggested byHemmat et al. (1994). However, homologies betweengroups became more evident with codominant mar-kers: microsatellites, minisatellite, SCARs, isozymes andthe 5S rDNA marker. Among the 12 linkage groupsidentified in the two parents, 10 pairs could clearly beassociated because of the presence of several commonmarkers. A complete merging of the two maps into onesingle consensus map could be accomplished only if theordering of markers were more precisely defined and ifcosegregation analysis were conducted in larger families.

Within the intraspecific F1

cross of Q. robur, a largenumber of markers (18% in both parental maps) exhib-ited segregation distortion regardless of the type ofmarker. In other examples of ‘‘pseudo-testcross’’ map-ping, distorted markers are present in lower propor-tions (9% in Eucalyptus urophylla, 8% in Eucalyptusgrandis Verhaegen and Plomion 1996, 15% in Pinuspalustris and 14% in Pinus elliottii Kubisiak et al.1995). As in pines, eucalypts and pedunculate oak, thereis a general trend towards a clustering of distortedmarkers on the linkage groups (linkage groups 2 and4 in the female map, and linkage groups 4 and 9 in themale map, Fig. 1).

The large number of distorted markers in Q. roburmay be related to the high genetic load observed in thisspecies. Interestingly, the variation of diversity amongeucalypts, pines and oaks as illustrated by the numberof loci being heterozygous in the two parents followsthe same pattern as the variation in the number of dis-torted loci. Although quantitative data on inbreedingdepression are not available in oaks, observations ofseedlings originating from control selfing crosses revealthat they experience several detrimental characters dur-ing their early development (poor germination, chloro-phyll deficiency, reduced growth) (Kleinschmit andKleinschmit 1996; and personal observations.).

Genetic map of Q. robur and other woody angiospermsand annual plants

To our knowledge this is the first reported genetic mapof an oak species and of any member of the Fagaceaefamily. Other linkage studies based on a reduced num-ber of isozyme loci and on several full-sib families haveindicated that 4 loci (Aap, ¸ap, Idh, Acp) are located on

the same linkage group in Q. robur (Zanetto et al. 1996)and Q. petraea (Muller-Starck et al. 1996). Interestinglythese 4 loci are distributed within linkage group 2 ofour genetic map (Fig. 1).

Genetic maps in other genera of broadleaved foresttrees are currently only available in Eucalyptus (Grat-tapaglia and Sederoff 1994; Verhaegen and Plomion1996; Byrne et al. 1995) and in Populus (Bradshaw et al.1994). The comparison with woody angiosperms istherefore extended to fruit trees having related speciesin natural forests and to a few annual plants for docu-mentary purposes (Table 3). With a few exceptions(Eucalyptus and Quercus) most existing maps of woodyangiosperms cover less than 80% of the genome. Basedon the data of Table 3, the physical genome size ofwoody angiosperms varies between 0.27 pg DNA/C(Prunus persica) and 0.9 pg DNA/C (Quercus robur),lying in the range of variation between Arabidopsis(0.15 pg DNA/C) and ¸ycopersicon esculentum (0.98 pgDNA/C) (Arumuganathan and Earle 1991). Mostwoody angiosperms have lower physical and geneticgenome sizes than annual plants. Quercus robur has thelargest physical genome among the angiosperm woodyspecies so far investigated, but its size is not greater thanthe genome of tomato. Curiously, the number of chro-mosomes and the genetic length of the genome in tom-ato and oak are also of a similar magnitude. Given that1 pg represents 0.915]109 bp (Bennet and Smith 1976),the physical distance per unit of genetic distance in Q.robur amounts to 0.77]106 bp/cM, which representsonly 3.21 times that of Arabidopsis thaliana (Table 3).

Microsatellites and consensus maps in oaksand Fagaceae

As illustrated in our example where two maps wereconstructed (Fig. 1), homologies between linkagegroups became obvious as codominant markers seg-regating in 1 : 2 : 1 or 1 : 1 : 1 : 1 or dominant markerssegregating in 3 : 1 could be mapped in both parents.Because the standard error of recombination fractionbetween markers segregating 1 : 1 : 1 : 1 (recoded 1 : 1)and markers segregating 1 : 1 is lower than for theothers (Ritter et al. 1990), their position on a linkagegroup is more precise. Hence, they are preferentialcandidates as anchor markers and for comparisonsacross maps.

Microsatellite primers are transportable across oakspecies as shown by the experiment conducted bySteinkellner et al. (1997b). Among the 18 microsatellitesmapped in Q. robur, which were originally developedfrom Q. petraea, polymerase chain reaction (PCR) am-plification and polymorphism were tested for 14 loci inother oak species and other genera of the Fagaceae. Allamplified products for the 14 loci were polymorphic inoak species belonging to the same botanical section(¸epidobalanus.) The number of successful loci was

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reduced to 64% in oak species of section Cerris and to47% in oak species of section Erythrobalanus. Theseresults are reinforced by our own experience withmicrosatellite loci developed from Quercus macrocarpa(Dow et al. 1995) and Quercus salicina (Kawahara T,unpublished data) and that were applied for mappingpurposes in the present contribution. However, suc-cessful amplification of oak microsatellites in relatedgenera of the family Fagaceae was reduced to 24% inFagus and to 47% in Castanea. Even though the pro-portion of amplified fragments and the level of poly-morphism decreased with increasing evolutionarydistance, microsatellites can be regarded as preferentialanchor and consensus markers across different oakspecies in the Quercus genus.

In the future these maps will be saturated usingadditional markers like amplified fragment lengthpolymorphism (AFLP, Vos et al. 1995) and furtherintegrated into a single consensus map when moremicrosatellites can be localised. This consensus mapwill be of great value in identifying quantitative traitloci of interest and particularly of those involved inadaptive traits of oaks.

Acknowledgements The authors gratefully acknowledge Dr. T.Kawahara for sharing primers of the MicJ-AG22 locus, AnneZanetto and Alexis Ducousso for their contribution in the controlledcross and Bernard Montousse for field technical assistance. Theresearch was supported by an EU research project (B104 CT96 0706)‘‘Development, optimization and validation of molecular tools forassessments of biodiversity in forest trees’’.

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